MCB Exam III Review Ji Woong Park December 13 th, 2014

Material Coverage My review covers 45 points. It’s lectures by Stewart, Huettner, Weihl, Amarasinghe, and Fremont. Due to the diverse range of topics (cancer to crystallography), you may need to use slides not in the review to help your understanding. But, the exam questions will be based on the review slides. There will be, however, some questions that require solid understanding/application to answer them (hence, the above point) Previous years’ exams will be helpful but it’s incorrect to assume they will be like Exam II.

Terms To Know for “Application” Questions RT-qPCR/RNAi/Knockout Gene or Mice Western Blot/IP/Co-IP Transfection/Infection Electrophysiology Mass Spec/NMR/Crystallography Site-Directed Mutagenesis Inhibitors/Dominant Negatives Fluorescence/Viability/Toxicity Assays

Telomere Function distinguishes between the chromosome end and a double strand break protects the chromosome from end-to-end fusions

a a a TRF2 TRF1 RAP1 TPP1 POT1 TIN2 TRF2 TRF1 TRF2 TRF1 An emerging paradigm: the telomere complex does not exclude DNA surveillance, repair and replication machinery; rather it directs, modulates and specializes the activities of these proteins to ensure high fidelity replication and telomere stability Old view DNA repair machinery New view DNA repair/replication machinery

The “telomere” hypothesis Population Doubling Time Telomere Length Senescence

The telomere hypothesis Telomere Length Time Stop Rbp53 Senescence

The telomere hypothesis Telomere Length Time Continued proliferation Rbp53 Crisis telomere dysfunction genetic catastrophe cell death

CAAUCCCAAUC Telomerase adds telomeric repeats to the 3’ termini of the chromosome hTERT hTR -only the catalytic (hTERT) and RNA (hTR) components are required for activity in vitro

The telomere hypothesis Telomere Length Time Senescence Crisis Stop Rbp53 1 in ~10 7 Stable telomere maintenance hTERT ALT

The telomere hypothesis Telomere Length Time Crisis Stop 1 in ~10 7 hTERT ALT

Telomerase +

Telomerase allows ALT cells to form tumors H-ras GM847 TUMORS hTERT Lacks telomerase Expresses SV40 Early region Immortal Stewart et al 2002

Extra-telomeric functions of hTERT Overexpression of mTERT in murine models increases tumor rates in aged mice Overexpression of hTERT results in resistance to apoptosis Telomerase is favored over ALT in human tumors Ectopic expression of mTERT in skin results “hairy” mice, increased stem cell pool hTERT expression is required in normal fibroblasts to avoid senescence

ECM Young fibroblast Altered ECM Senescent fibroblast Immune cell Senescent epithelial cell Cancer cell Preneoplastic cell Epithelial cell Endothelial cell Senescence evasion Tumor Senescence Normal Stromal Promotion Premalignant Time/Stress

For additional reading (highly recommend)

Types of Stem Cells Embryonic – from the inner cell mass of pre- implantation embryos, prior to formation of the 3 germ layers (ectoderm, mesoderm, endoderm) Somatic – undifferentiated cells found in specific locations in “mature” tissues iPS cells – induced pluripotent stem cells generated by reprogramming differentiated cells (or cell nuclei, i.e. therapeutic cloning)

Reprogramming SCNT – somatic cell nuclear transfer (reproductive and therapeutic cloning) – deterministic and fairly rapid iPS – induced pluripotent stem cells – slow and stochastic (until recently) Transdifferentiation – conversion of one terminally differentiated cell type into another without de- differentiation to an immature phenotype. Must rule out cell fusion or other explanations.

Generating iPS cells Express transcription factors: Oct3/4, Sox2, Klf4 and c-Myc (OSKM) OR Oct3/4, Sox2, Nanog and Lin28 Initial de-differentiation and proliferation (day 1-3, enhanced by Myc); histone modification and chromatin reorganization 2 nd wave of gene expression - stem cell and development related genes (day 9-12); DNA demethylation and X reactivation

Transdifferentiation Conversion from one differentiated cell type to another without evident de-differentiation and re-differentiation Must not be confused by cell fusion or selection for rare pluripotent cells in the source material. Induced by expression of transcription factors and microRNAs

Protein Degradation in the Cell UPS Aggresome Autophagy Endocytosis Nucleus Ub

Protein Degradation  Ubiquitin/Proteasome Pathway 80-90% Most intracellular proteins Lysosomal processes 10-20% Extracellular proteins Cell organelles Some intracellular proteins

UBIQUITINK G  Small peptide that is a “TAG”  76 amino acids  C-terminal glycine - isopeptide bond with the  -amino group of lysine residues on the substrate  Attached as monoubiquitin or polyubiquitin chains

Ubiquitination of proteins is a FOUR-step process  First, Ubiquitin is activated by forming a link to “enzyme 1” (E1).  Then, ubiquitin is transferred to one of several types of “enzyme 2” (E2).  Then, “enzyme 3” (E3) catalizes the transfer of ubiquitin from E2 to a Lys  -amino group of the “condemned” protein.  Lastly, molecules of Ubiquitin are commonly conjugated to the protein to be degraded by E3s & E4s AMP

The proteasomal DUB Usp14 impairs protein degradation Lee, BH et al Nature 467:

Autophagy Lysosomal degradation of proteins and organelles Occurs via three routes – Macroautophagy – Microautophagy (direct uptake of cellular debris via the lysosome) – Chaperone mediated autophagy (selective import of substrates via Hsc70 and Lamp2a)

Selective Autophagy Aggregaphagy– p62/SQSTM1, Nbr1 Mitophagy – Parkin, Nix Reticulophagy – endoplasmic reticulum Ribophagy – translating ribosomes Xenophagy – e.g. Salmonella via optineurin Lipophagy – autophagy mediated lipolysis Performed by an expanding group of ubiquitin adaptors

Rapamycin as an inducer of autophagy  Immunosuppressant used to treat transplant rejection  Inhibits the mTOR pathway  mTOR integrates extrinsic growth signals and cellular nutrient status and energy state  Active mTOR  Protein synthesis and cell growth  Inactive mTOR (or rapamycin treatment)  Inhibition of protein synthesis and increased autophagic degradation of protein

Protein Structures from an NMR Perspective Distance from Correct Structure NMR Data Analysis Correct structure X Not A Direct Path! Interpreting NMR Data Requires Making Informed “Guesses” to Move Toward the “Correct” Fold Initial rapid convergence to approximate correct fold Iterative “guesses” allow “correct” fold to emerge Analyzing NMR Data is a Non-Trivial Task! there is an abundance of data that needs to be interpreted

Protein Structure Determination by NMR Stage I—Sequence specific resonance assignment State II – Conformational restraints Stage III – Calculate and refine structure

Why use deuteration? What are the advantages? What are the disadvantages?

4.1Å 2.9Å NOE CHCHCHCH NH NH CHCHCHCH J NOE - a through space correlation (<5Å) - distance constraint Coupling Constant (J) - through bond correlation - dihedral angle constraint Chemical Shift - very sensitive to local changes in environment in environment - dihedral angle constraint Dipolar coupling constants (D) - bond vector orientation relative to magnetic field to magnetic field - alignment with bicelles or viruses D NMR Structure Determination

Analysis of the Quality of NMR Protein Structures Is the “Average” NMR Structure a Real Structure? No-it is a distorted structure  level of distortions depends on the similarity between the structures in the ensemble  provides a means to measure the variability in atom positions between an ensemble of structures Expanded View of an “Average” Structure Some very long, stretched bonds Position of atoms are so scrambled the graphics program does not know which atoms to draw bonds between Some regions of the structure can appear relatively normal

An 7-step program for protein structure determination by x-ray crystallography 1. Produce monodisperse protein either alone or as relevant complexes 2. Grow and characterize crystals 3. Collect X-ray diffraction data 4. Solve the phase problem either experimentally or computationally 5. Build and refine an atomic model using the electron density map 6. Validation: How do you know if a crystal structure is right? 7. Develop structure-based hypothesis

1.Produce monodisperse protein either alone or as relevant complexes Methods to determine protein purity, heterogeneity, and monodispersity  Gel electrophoresis (native, isoelectric focusing, and SDS-PAGE)  Size exclusion chromatography  Dynamic light scattering  Circular Dichroism Spectroscopy Characterize your protein using a number of biophysical methods Establish the binding stoichiometry of interacting partners

2. Grow and characterize crystals  Hanging Drop vapor diffusion  Sitting drop, dialysis, or under oil  Macro-seeding or micro-seeding  Sparse matrix screening methods  Random thinking processes, talisman, and luck The optimum conditions for crystal nucleation are not necessarily the optimum for diffraction-quality crystal growth Space Group P2 1 4 M3 /ASU diffraction >2.3Å 14.4% Peg6K NaCacodylate pH mM CaCl 2 Space Group P M3 + 3 MCP-1/ASU diffraction > 2.3Å 18% Peg4K NaAcetate pH mM MgCl 2 Space Group C2 2 M3 /ASU diffraction >2.1Å 18% Peg4K Malic Acid/Imidazole pH mM CaCl 2 Commercial screening kits available from Hanging DropSitting drop

No Xtals? Decrease protein heterogeneity  Remove purification tags and other artifacts of protein production  Remove carbohydrate residues or consensus sites (i.e., N-x-S/T)  Determine domain boundaries by limited proteolysis followed by mass spectrometry or amino-terminal sequencing. Make new expression constructs if necessary.  Think about the biochemistry of the system! Does your protein have co- factors, accessory proteins, or interacting partners to prepare as complexes? Is their an inhibitor available? Are kinases or phosphatases available that will allow for the preparation of a homogeneous sample?  Get a better talisman

3. Collect X-ray diffraction data  Initiate experiments using home-source x-ray generator and detector  Determine liquid nitrogen cryo-protection conditions to reduce crystal decay  While home x-rays are sufficient for some questions, synchrotron radiation is preferred  Anywhere from one to hundreds of crystals and diffraction experiments may be required Argonne National Laboratory Structural Biology Center beamlineID19 at the Advanced Photon Source

4. Solve the phase problem either experimentally or computationally  Structure factor equation:  By Fourier transform we can obtain the electron density. We know the structure factor amplitudes after successful data collection. Unfortunately, conventional x-ray diffraction doesn’t allow for direct phase measurement. This is know as the crystallographic phase problem.  Luckily, there are a few tricks that can be used to obtain estimates of the phase  (h,k,l) Experimental Phasing Methods  MIR - multiple isomorphous replacement - need heavy atom incorporation  MAD - multiple anomalous dispersion- typically done with SeMet replacement  MIRAS - multiple isomorphous replacement with anomalous signal  SIRAS - single isomorphous replacement with anomalous signal Computational Methods  MR - molecular replacement - need related structure  Direct and Ab Initio methods - not yet useful for most protein crystals

MAD phasing statistics for the AP-2  -appendage

Electron density for the AP-2  appendage Initial bones trace for the AP-2  appendage Final trace for the AP-2  appendage 5. Build an atomic model using the electron density map

The resolution of the electron-density map and the amount of detail that can be seen ResolutionStructural Features Observed 5.0 ÅOverall shape of the molecule 3.5 ÅCa trace 3.0 ÅSide chains 2.8 ÅCarbonyl oxygens (bulges) 2.5 ÅSide chain well resolved, Peptide bond plane resolved 1.5 ÅHoles in Phe, Tyr rings 0.8 ÅCurrent limit for best protein crystals

6. Validation: How do you know if a crystal structure is right? The R-factor R =  (|Fo-Fc|)/  (Fo) where Fo is the observed structure factor amplitude and Fc is calculated using the atomic model. R-free An unbiased, cross-validation of the R-factor. The R-free value is calculated with typically 5-10% of the observed reflections which are set aside from atomic refinement calculations. Main-chain torsions: the Ramachandran plot Geometric Distortions in bond lengths and angles Favorable van der Waals packing interactions Chemical environment of individual amino acids Location of insertion and deletion positions in related sequences

Structure-Based Mutagenesis of the  -appendage 7. Develop structure-based hypothesis

Selection of E16 specific epitope variants of DIII Yeast library of DIII variants created by error prone PCR DIII mutations at Ser 306, Lys 307, Thr E330 and Thr 332 significantly diminish E16 binding Pooled DIII mAbs E16 staining E -DIII

GOOD LUCK – Interview season is coming!